The cardiac Na/Ca exchanger (NCX) is the principal Ca efflux mechanism in cardiac myocytes and plays an essential role in regulating cardiac contractility. Characterizing the exchanger's regulatory properties is important for understanding both normal cardiac physiology and the perturbations that occur in pathogenic conditions. This project focuses on the allosteric regulation of NCX activity by cytosolic Ca and its modulation by the cytoskeleton. The hypothesis guiding these studies is that allosteric Ca activation displays hysteresis so that activity becomes Ca-independent following NCXactivation (persistent Ca activation). We propose that the hysteresis is due to (a) cytoskeletal interactions involving the "beta repeat regions" of the NCX protein that restrict access of Ca to the regulatory binding sites and (b) interaction of PIP2 with the so- called XIP region of the NCX protein. The project has 5 specific aims directed toward the following goals: (1) Elevations in cytosolic [Na] induce a Ca-independent mode of NCXactivity. We will measure the [Na] dependence of this response and investigate the role of cytoskeletal interactions, PIP2 and XIP mutations in the development of this activity. (2) We will determine whether stimulation of F-actin dynamics by protein kinase C, NO donors or activated Rac or Cdc42 enhances the development of Ca-independent activity and accelerates the rate of allosteric Ca activation. (3) We will determine how cytoskeletal interactions, cytosolic [Na] and PIP2 influence the development and relaxation of persistent Ca activation. (4) Mutations will be created in the beta repeat regions of the exchanger and their influence on cytoskeletal interactions, allosteric Ca activation and Ca-independent NCX activity will be assessed. (5) We will determine whether cytoskeletal interactions modulate NCX activity In rat neonatal and adult cardiac myocytes. Transfected CHO cells will be used in Aims 1-4. Digital imaging techniques using fluorescent Ca probes will be used to measureNCX transport activity.